Analysis of mitotic microtubule-associated proteins

using mass spectrometry identifies astrin, a

spindle-associated protein
Gary J. Mack and Duane A. Compton*
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755

Edited by J. Richard McIntosh, University of Colorado, Boulder, CO, and approved October 17, 2001 (received for review July 18, 2001)

We purified microtubules from a mammalian mitotic extract and somes. Thus, we have generated a comprehensive list of all major
obtained an amino acid sequence from each microtubule-associ- motor and nonmotor mitotic microtubule-associated proteins.
ated protein by using mass spectrometry. Most of these proteins
are known spindle-associated components with essential func- Materials and Methods
tional roles in spindle organization. We generated antibodies Cell Culture. HeLa cells were maintained in DMEM containing
against a protein identified in this collection and refer to it as astrin 10% FCS, 50 units兾ml penicillin, and 50 ␮g兾ml streptomycin.
because of its association with astral microtubule arrays assembled CFPAC-1 cells were maintained in Iscove’s modified Dulbecco’s
in vitro. Astrin is ⬇134 kDa, and except for a large predicted medium containing 10% FCS, 50 units兾ml penicillin, and 50
coiled-coil domain in its C-terminal region it lacks any known ␮g兾ml streptomycin. Cells were grown at 37°C in a humidified
functional motifs. Astrin associates with spindle microtubules as incubator with a 5% C02 atmosphere.
early as prophase where it concentrates at spindle poles. It localizes
throughout the spindle in metaphase and anaphase and associates Microtubule Aster Preparation and Enrichment. Mitotic extracts and
with midzone microtubules in anaphase and telophase. Astrin also microtubule asters were prepared as described (22). Latrunculin
localizes to kinetochores but only on those chromosomes that have B (5 ␮g兾ml) was added to the final extract to reduce actin
congressed. Deletion analysis indicates that astrin’s primary spin- polymerization and the contamination of microtubule pellets
dle-targeting domain is at the C terminus, although a secondary with actin and actin-associated proteins. Extracts corresponding
domain in the N terminus can target some of the protein to spindle to ⬇3 ⫻ 107 cells were layered onto a 50% (wt兾vol) sucrose
poles. Thus, we have generated a comprehensive list of major cushion in KHM buffer (78 mM KCL兾50 mM Hepes, pH 7.0兾4
mitotic microtubule-associated proteins, among which is astrin, a mM MgCl2兾2 mM EGTA兾1 mM DTT), and microtubules were
nonmotor spindle protein. collected by sedimentation at 100,000 ⫻ g for 2 h at 4°C. The
insoluble pellet was collected directly in SDS兾PAGE sample

T he spindle is a complex microtubule-based superstructure

responsible for chromosome segregation in mitosis and
meiosis (1–6). Spindle microtubules are organized in a complex
buffer. The inclusion of Latrunculin B and the use of 50%
sucrose cushions significantly reduced contamination of the
insoluble pellet by nonmicrotubule-binding proteins relative to
process that requires both motor and nonmotor microtubule- the initial aster enrichment reported previously (23).
associated proteins. A great deal of emphasis has been placed on Peptide sequencing was performed at the Harvard Micro-
the functional contribution that motor proteins make to spindle chemistry Facility (Cambridge, MA) by microcapillary reverse-
organization because of their inherent biological activity. The phase high pressure liquid chromatography nanoelectrospray
strong sequence homology of the conserved motor domain in tandem mass spectrometry on a Finnigan LCQ quadrupole ion
these proteins has facilitated both their rapid identification and trap mass spectrometer.
characterization during spindle assembly. These investigations
have demonstrated that motors of both the kinesin and dynein Antibodies. The N-terminal 609 aa of astrin was expressed in
families play important roles in such varied aspects of spindle bacteria as a fusion protein with glutathione S-transferase by
organization as spindle bipolarity (6), chromosome movement inserting a 1,827-nt segment of astrin cDNA into the SmaI and
(7–10), checkpoint regulation (11, 12), spindle pole organization EcoRI sites of pGEX-5X-2 (Amersham Pharmacia). The C-
(13–16), and the regulation of microtubule dynamics (17). In
terminal 586 aa of astrin was expressed in bacteria as a fusion
contrast, the functional contribution that nonmotor proteins
protein with glutathione S-transferase by inserting a 1,951-nt
make to spindle organization has not progressed as rapidly. In
segment of astrin cDNA into the EcoRI site of pGEX-5X-1.
part, the lag in progress in defining the roles of nonmotor spindle
Glutathione S-transferase-astrin fusion proteins were induced
proteins stems from the lack of clearly defined biological activity
with 1 mM isopropyl ␤-D-thiogalactoside in liquid cultures, and
of nonmotor proteins. A further complication arises from the
lack of any clear homology among nonmotor proteins to facil- recombinant proteins were purified by electroelution from SDS兾
itate their identification. Thus, although some nonmotor spindle PAGE and injected into rabbits (Covance Research Products,
proteins have been characterized (18, 19), others are less well Denver, PA).
understood (20, 21), and many others may await identification. Antibodies against NuMA (22), HSET (24), Eg5 (24), Arp1
In this article we use mass spectrometry to identify the
proteins associated with microtubules assembled in a cell-free This paper was submitted directly (Track II) to the PNAS office.
mitotic extract under conditions where ⬎90% of microtubules
Abbreviation: GFP, green fluorescent protein.
are organized into astral arrays. Most proteins identified through
Data deposition: The sequence reported in this paper has been deposited in the GenBank
this strategy have known functional roles for spindle assembly. A database (accession no. AF399910).
protein identified in this collection is referred to as astrin
*To whom reprint requests should be addressed. E-mail: duane.a.compton@
because of its association with microtubule asters assembled dartmouth.edu.
under these in vitro conditions. Astrin is a nonmotor coiled coil The publication costs of this article were defrayed in part by page charge payment. This
containing protein that associates with spindles throughout article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
mitosis and localizes to kinetochores of congressed chromo- §1734 solely to indicate this fact.

14434 –14439 兩 PNAS 兩 December 4, 2001 兩 vol. 98 兩 no. 25 www.pnas.org兾cgi兾doi兾10.1073兾pnas.261371298

(25), tubulin (DM1A, Sigma–Aldrich), dynein (26), and TOGp insoluble pellet was run in parallel. Immunoblotting for NuMA
(23) have all been described previously. Antibodies against KIF4 and Eg5, two proteins known to associate with microtubules
and MCAK both were generated by immunizing rabbits with under these conditions, verified the enrichment of microtubules
recombinant proteins expressed in bacteria (A. Levesque and relative to the total extract by using this technique (Fig. 1 A).
D.A.C., unpublished results). Antibodies against TACC3, Sixteen protein bands were enriched in the extract after
MAP4, and centromeres were generous gifts from Jordan Raff, microtubule polymerization compared with the control sample
Chloe Bulinski, and Kevin Sullivan, respectively. in which microtubules were not polymerized because of the
absence of taxol. Each protein band was excised from the
Immunologic Techniques. Indirect immunofluorescence micros- polyacrylamide gel and amino acid sequence obtained from each
copy of HeLa and CFPAC-1 cells was performed as described band by using mass spectrometry of tryptic peptides. Although
(27). Immunoblotting of cell extracts or total cell protein was some stained bands contained more than one major protein of
performed as described (27). equal size, all these proteins were represented in the genome
database, and we classified them into five categories (Fig. 1 A).
Astrin cDNA Analysis and Transfection Constructs. Full-length astrin The first category includes NuMA, TOGp, p150Glued, Eg5,
cDNA in the vector pCMVSPORT 6 (GenBank accession no. HSET, and tubulin and are proteins with previously identified
AL530294) was purchased from Invitrogen and designated roles in both spindle organization in vivo and microtubule aster
pSPORT-Astrin. Protein sequence alignment and identity organization in vitro. The second category includes KIAA0622
shading was preformed by using MEGALIGN of DNAstar (the human homologue of the fruit fly gene orbit), KIF4,
(Madison, WI). TACC3, TPX2, and MCAK and are proteins with established
Expression constructs were created in the vector series spindle functions in other systems but are untested for a func-
pEGFP-C (CLONTECH) with the exception of construct H, tional role in the organization of microtubules into asters in this
which used the vector pEGFP-N1. Fragment A was amplified system. Immunodepletion of KIF4 or MCAK did not alter
from pSPORT-Astrin by PCR using Pfu polymerase (Strat- microtubule aster organization in this system in any detectable
agene) and the primers AstF (5⬘-ATGTGGCGAGT- manner (A. Levesque and D.A.C., unpublished results), sub-
GAAAAAACTGAGCC-3⬘) and AstR (5⬘-GCTCAGAAAT- stantiating our previous conclusion that cytoplasmic dynein,
TCCAGCAATCCC-3⬘) and inserted into the Ecl136II site of Eg5, and HSET may be the primary microtubule motors respon-
pEGFP-C3. Fragment B was amplified by using the primers AstF sible for organizing microtubule asters in this system (24). The
and AstR2 (5⬘-GTTTGTCCACCTCCTGAGAGAGCC-3⬘) and third category includes the microtubule-associated proteins
inserted into the Ecl136II site of pEGFP-C3. Fragment C was MAP4 and MAP7, which were expected to have been enriched
amplified by using the primers AstF and AstR and digested with given the isolation strategy used. The fourth category includes
EcoRI. The resulting 1,827-nt fragment was inserted into the plectin, clathrin, SMC1 and -3, and HSP70 and -71, which have
Ecl136II兾EcoRI site of pEGFP-C3. Fragment D was amplified no documented role as microtubule-associated proteins and may
by using the primers AstF and AstR and digested with PvuII. The represent contaminants. The final category includes a protein of
resulting 1,374-nt fragment was inserted into the Ecl136II site of ⬇140 kDa that we refer to as astrin on the basis of its enrichment
pEGFP-C1. Fragment E was generated by digesting pSPORT- with microtubules under conditions where most microtubules
Astrin with DraI and inserting the resulting 3,207-nt fragment assemble into asters.
into the Ecl136II site of pEGFP-C1. Fragment F was generated To verify the mass spectrometry data we performed immu-
by digestion of pSPORT-Astrin with EcoRI and DraI and noblot analysis of the 10,000 ⫻ g-soluble and -insoluble protein
inserting the resulting 1,865-nt fragment into the EcoRI兾SmaI fractions obtained after microtubule assembly in these mitotic
site of pEGFP-C3. Fragment G was generated by digesting extracts (Fig. 1B). All the proteins identified by mass spectrom-
pSPORT-Astrin with SstI and DraI and inserting the resulting etry for which we had access to antibodies displayed enrichment
492-nt fragment into the SacI兾SmaI site of pEGFP-C3. Frag- in the microtubule pellet fraction with some (e.g., TOGp) being
ment H was generated by digestion of pSPORT-Astrin with SacI highly enriched, others (e.g., Eg5) moderately enriched, and yet
and inserting the resulting 1,810-nt fragment into the SacI site of others [e.g., dynactin(Arp1)] weakly enriched. Cytoplasmic dy-
pEGFP-N1. nein provides an example of a protein that remains largely
soluble and shows very little enrichment on microtubules under
Transient Transfection. HeLa cells were seeded onto glass cover- these conditions.
slips and grown to 60–80% confluency. Cells were transfected
with 2 ␮g of plasmid DNA and 8 ␮l of Lipofectamine reagent Astrin Is a Spindle-Associated Protein. To characterize astrin, the
(GIBCO兾BRL) in 1 ml of Opti-MEM (GIBCO兾BRL) accord- only previously unidentified protein identified in this collection,
ing to manufacturer recommendations. The transfection reagent we obtained a full-length cDNA encoding this protein from
was removed after 3 h, replaced with complete growth medium, Invitrogen. Sequence analysis indicates that the cDNA is
and processed for indirect immunofluorescence by using anti- 3,843-nt long and encodes a protein of 1,193 aa with a predicted
green fluorescent protein (GFP) antibody after 24 h (Molecular molecular mass of 134,400 Da (Fig. 2; accession no. AF399910).
Probes). With the exception of two predicted coiled-coil domains in the
CELL BIOLOGY

C-terminal half of the protein and numerous predicted phos-

Results phorylation sites, astrin contains no identifiable functional
Identification of the Major Mitotic Microtubule-Associated Proteins motifs. BLAST searches identified numerous expressed sequence
Using Mass Spectrometry. Microtubules were polymerized in a tag sequences and cDNA clones encoding partial segments of
cell-free mitotic extract under conditions where ⬎90% of mi- astrin, only one of which had the correct ORF (accession no.
crotubules organized into astral arrays (22). Microtubule com- BC000322). Astrin also showed strong homology to proteins
plexes were separated from soluble extract components by from mouse and rat (Fig. 2). A partial sequence of a mouse gene
sedimentation through 50% sucrose. The microtubule pellet was of unknown function called S17 (28) showed 56.4% amino acid
solubilized in SDS, and the proteins were identified by Coomas- identity to the N-terminal 711 aa of astrin. The C-terminal 397
sie blue staining after size separation on 7.5% SDS兾PAGE (Fig. aa of astrin were 72.8% identical to the rat protein Spag5, which
1A). As a control for the microtubule-dependent enrichment of was identified in a yeast two-hybrid screen for proteins that
proteins, an equivalent amount of mitotic extract was incubated interact with Odf1, an outer dense filament protein from sperm
in the absence of taxol, and a proportional quantity of that flagella (29).

Mack and Compton PNAS 兩 December 4, 2001 兩 vol. 98 兩 no. 25 兩 14435

 Fig. 2. Alignment of the human astrin protein sequence with mouse S17 and
rat Spag5 protein sequences. Identical amino acids residues are shaded, and
predicted coiled-coil domains spanning amino acids 550 – 857 and 970 –1,175
are in boxes.

We raised rabbit polyclonal antibodies against the N-terminal

609 aa and the C-terminal 586 aa of astrin. Both antibodies were
species-specific and reacted specifically with a protein of ⬇140
kDa on immunoblots of total HeLa cell protein (Fig. 1B and data
not shown). Both antibodies showed similar staining patterns in
human cells under various fixation conditions (Fig. 3). Astrin was
diffuse in the cytoplasm of interphase cells with some concen-
trated near the centrosome (Fig. 3A). In prophase, astrin asso-
ciated with microtubules and concentrated at the vertices of the
developing spindle poles (Fig. 3B). Astrin localized to spindle
microtubules throughout prometaphase, metaphase, anaphase,
and telophase (Fig. 3 C–F). In metaphase, astrin localized
throughout the central spindle in a pattern that mirrored mi-
crotubules. It also associated with midzone microtubules in
anaphase and telophase. The localization of astrin to the spindle
during mitosis was microtubule-dependent as the staining pat-
tern was abolished by nocodazole treatment.

Fig. 1. Identification of mitotic microtubule-associated proteins. (A) Equiv-

alent quantities of the total extract and the insoluble fractions obtained after
incubation with (⫹) and without (⫺) taxol were separated by size on 7.5%
SDS兾PAGE and stained with Coomassie blue. Protein identities revealed by
mass spectrometry are indicated on the right of each protein band. The lower
panels show immunoblots of each of these three samples for NuMA and Eg5
to verify the enrichment of microtubule-associated proteins by using this
technique. Molecular masses in kDa are indicated on the left. (B) Immunoblots
of various microtubule-associated proteins (as indicated) identified by mass
spectrometry of soluble (S) and insoluble (P) fractions obtained after micro-
tubule aster assembly in the mitotic extract.

14436 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.261371298 Mack and Compton

 Fig. 4. Localization of astrin to kinetochores. CFPAC-1 cells were fixed in
glutaraldehyde and stained with antibodies against astrin, anti-centromere
antibodies (ACA), and the DNA-specific dye 4⬘,6-diamidino-2-phenylindole
(DAPI) as indicated. Cells were classified as being in prometaphase (A) and
metaphase (B) on the basis of chromosome alignment. Arrowheads highlight
sister kinetochores in metaphase that stain for astrin, and the arrow highlights
the kinetochore of an unaligned chromosome in prometaphase that lacks
astrin staining. (Bar, 10 ␮m.)

To map the domain of astrin required for spindle targeting, we

expressed various segments of astrin in HeLa cells and deter-
mined the localization of each segment during mitosis (Fig. 5A).
Immunoblot analysis of cell extracts obtained after transient
expression of each domain confirmed that a protein of the
expected size was expressed from each plasmid (Fig. 5B).
Full-length astrin and segments of astrin lacking short parts of
either the N or C termini localized throughout the mitotic
spindle in metaphase in a pattern indistinguishable from the
endogenous protein (Fig. 5C, A, B, and E). Central segments of
astrin containing the predicted coiled-coil regions displayed
spindle localization similar to the full-length protein, although
the spindle staining intensity was diminished and there was
higher cytoplasmic background (Fig. 5C, F and H). The extreme
C-terminal 129 aa failed to localize to the spindle and was
localized diffusely in mitotic cells (Fig. 5C, G). Finally, expres-
sion of the N-terminal region lacking the predicted coiled-coil
domains showed diffuse cytoplasmic staining in addition to some
spindle staining (Fig. 5C, C and D). The spindle staining
observed with these N-terminal fragments was dissimilar to the
full-length protein, because they were restricted to spindle poles
and did not extend throughout the spindle. Kinetochore staining
was not observed consistently under conditions of transient
expression, thus we were unable to define unequivocally a
domain of astrin responsible for kinetochore accumulation.
Fig. 3. Cell cycle distribution of astrin. HeLa cells were fixed in cold methanol Discussion
and stained with antibodies against astrin, tubulin, and the DNA-specific dye
We report the identification of all major proteins associated with
4⬘,6-diamidino-2-phenylindole (DAPI) as indicated. Cells were classified as
being in interphase (A), prophase (B), prometaphase (C), metaphase (D), microtubules assembled in a mammalian mitotic extract. The use
anaphase (E), and telophase (F) on the basis of the morphology and alignment of mass spectrometry to sequence each protein is unbiased to the
CELL BIOLOGY

of chromosomes and spindle organization. (Bar, 10 ␮m.) specific biological function of any given protein and succeeded
in identification of both motor and nonmotor microtubule-
associated proteins, most of which have known functional roles
We also observed a punctate staining pattern for astrin in spindle organization. These results suggest that these proteins
adjacent to the chromosomes during metaphase reminiscent of represent a comprehensive catalogue of all major noncentroso-
kinetochores, and double labeling with an anti-centromere an- mal and nonchromosomal spindle-associated proteins. Addi-
tibody verified that astrin concentrates at kinetochores during
metaphase (Fig. 4B). Surprisingly, in prometaphase, kineto- tional experimental approaches will be needed to identify those
chores of chromosomes that were unaligned did not stain for proteins that associate only transiently with asters兾spindles (such
astrin, whereas those chromosomes that were aligned at the as cytoplasmic dynein) and those proteins that are minor com-
metaphase plate displayed kinetochore staining (Fig. 4A). Astrin ponents of asters兾spindles.
required intact microtubules for both its establishment and Among this collection of mitotic microtubule-associated pro-
maintenance at kinetochores, because nocodazole treatment teins is astrin, a coiled coil-containing, nonmotor protein that
abolished all kinetochore staining. localizes throughout the spindle during mitosis. Astrin is unique

Mack and Compton PNAS 兩 December 4, 2001 兩 vol. 98 兩 no. 25 兩 14437

Fig. 5. Expression of GFP-astrin fusion proteins in HeLa cells. (A) Schematic diagram of astrin fragments that were tested for their ability to bind the mitotic
spindle. ⫹⫹⫹, strong binding; ⫹⫹, moderate binding; ⫹, weak binding; ⫺, no binding; shaded boxes, regions of astrin predicted to form a coiled-coil structure.
(B) Immunoblot of transfected HeLa cell lysates expressing the various GFP-astrin fusion proteins. Molecular masses in kDa are indicated on the left. (C)
Immunofluorescent images of metaphase mitotic cells expressing the various GFP-astrin fusion proteins. Cells were fixed in cold methanol, and GFP was detected
with a rabbit anti-GFP antibody. (Bar, 10 ␮m.)

in that although it is expressed throughout the cell cycle, it is useful marker for kinetochore maturation and chromosome
localized only to microtubules during mitosis. The C terminus is alignment.
the primary determinant for targeting astrin to spindles; how- Currently, the function of astrin is unknown. Astrin’s specific
ever, the association of astrin with spindles may involve more association with spindles would suggest a functional role in
complex intermolecular interactions, because the N-terminal spindle organization; however, we have been unable to confirm兾
domain is sufficient to target some of the protein to spindle refute that suggestion, because our antibodies do not react
poles. Another unusual aspect of astrin is its association with efficiently with the native protein, making immunodepletion of
kinetochores. It is the only protein to our knowledge that mitotic extracts and microinjection experiments uninformative.
associates with kinetochores of only those chromosomes that We speculate that astrin may play a structural role within the
have aligned at the metaphase plate. Thus, although astrin spindle based on two observations. First, the targeting of astrin
requires intact microtubules to localize to kinetochores and is to spindles is complex and involves both the N and C termini,
thus not a constitutive kinetochore component, it can serve as a indicating that astrin may bind to multiple spindle components

14438 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.261371298 Mack and Compton

through different domains. Second, astrin is homologous to rat in microtubule anchoring to the centrosome (31). Thus, by
Spag5, a gene product that was identified as interacting with odf1 analogy astrin may play a structural role in the spindle by
(outer dense filament protein 1) from sperm (29). The outer cross-linking microtubules to other spindle components.
dense filament is postulated to play a role in sperm motility or
elastic recoil through cross-links with axonemal microtubules We thank Kevin Sullivan (Scripps Research Institute), Chloe Bulinski
(30). Another component of the outer dense filament (odf2) has (Columbia University) and Jordan Raff (Wellcome兾Cancer Research
been shown recently to localize preferentially to the mother Campaign Institute) for generously providing antibodies. This work was
centriole in centrosomes, where it was speculated to play a role supported by National Institutes of Health Grant GM51542.

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Mack and Compton PNAS 兩 December 4, 2001 兩 vol. 98 兩 no. 25 兩 14439